Topical composition for skin

ABSTRACT

A topical skin composition containing a dipeptide compound represented by, formula (1) or a salt of the dipeptide:                    
     wherein R 1  represents a, hydrogen atom, an alkyl group, an alkanoyl group, or —CH(R 6 )COOR 7  (wherein R 6  represents a hydrogen atom or a lower alkyl group, and R 7  represents a hydrogen atom, a lower alkyl group, a lower alkenyl group, or an aralkyl group); 
     R 2  represents a hydrogen atom or an alkyl group which may have a substituent; 
     R 3  represents a lower alkyl group or a phenyl group; 
     R 4  represents a hydrogen atom or a lower alkyl group, and may form a heterocyclic ring together with R 5  and an adjacent nitrogen atom; 
     R 5  represents a hydrogen atom, an alkyl group which may have a substituent, or an aralkyl group which may have a substituent, and may form the heterocyclic ring together with R 4 ; 
     X represents —COOR 8  (wherein R 8  represents a hydrogen atom, a lower alkyl group, a lower alkenyl group, or an aralkyl group) or —SO 3 H; and 
     n is an integer of 0-4; and a pharmaceutically acceptable carrier therefor.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a topical composition for the skin(hereinafter may be simply referred to as a topical skin composition)exhibiting effects of retarding aging of the skin or rejuvenating theskin and of suppressing hair growth.

2. Background Art

Studies have revealed that aging of the skin is mainly caused byadvancing age, drying, oxidation, or sunlight (i.e., UV rays). Aging ofthe skin is recognized by a decrease in collagen or elastin in thedermis of the skin; a decrease in mucopolysaccharides, includinghyaluronic acid; or the presence of cells which are damaged by UV rays.

For the prevention of wrinkle formation, however, sufficient effectshave not yet been attained, for example, by a collagen-containingcosmetic composition. In addition, a number of research projects havefocused on skin aging caused by exposure to UV rays. However, cosmeticcompositions superseding UV absorbing agents or UV protecting agentshave not yet been developed.

There is a trend towards a preference for hairless bodies, particularly,hairless arms or legs, for reasons of aesthetic appearance. Variousmethods have been tried in the pursuit of body hair removal, forexample, a mechanical hair-removal method making use of a shaver orhair-tweezers, a method for removing body hair from the hair root by useof a hair removing agent, and a method for removing body hair throughchemical action of a hair removing agent.

However, the aforementioned hair removing methods physically orchemically stimulate the skin, and are limited by the unsatisfactoryduration of the hairless state. Therefore, there is a need for thedevelopment of a method which facilitates removal of body hair.

SUMMARY OF THE INVENTION

In view of the foregoing, an object of the present invention is toprovide a topical composition for the skin exhibiting effects withrespect to retarding aging of the skin, such as wrinkle formation, orrejuvenating the skin, and of suppressing hair growth.

The present inventors have found that a dipeptide compound having aspecific structure or a salt thereof exhibits excellent effects withrespect to suppressing wrinkle formation, preventing a reduction in skinelasticity, and suppressing hair growth, and that the compound or thesalt is effectively employed in a topical skin composition for retardingaging of the skin or suppressing hair growth.

Accordingly, the present invention provides a topical skin compositioncomprising a dipeptide compound represented by formula (1) or a salt ofthe dipeptide:

wherein R¹ represents a hydrogen atom, an alkyl group, an alkanoylgroup, or —CH(R⁶)COOR⁷ (wherein R⁶ represents a hydrogen atom or a loweralkyl group, and R⁷ represents a hydrogen atom, a lower alkyl group, alower alkenyl group, or an aralkyl group);

R² represents a hydrogen atom or an alkyl group which may have asubstituent;

R³ represents a lower alkyl group or a phenyl group;

R⁴ represents a hydrogen atom or a lower alkyl group, and may form aheterocyclic ring together with R⁵ and an adjacent nitrogen atom;

R⁵ represents a hydrogen atom, an alkyl group which may have asubstituent, or an aralkyl group which may have a substituent, and mayform the heterocyclic ring together with R⁴;

X represents —COOR⁸ (wherein R⁸ represents a hydrogen atom, a loweralkyl group, a lower alkenyl group, or an aralkyl group) or —SO₃H; and

n is an integer of 0-4; and a pharmaceutically acceptable carriertherefor.

The present invention also provides, as a novel compound which has notyet been described in any literature,N-(carboxymethyl)phenylalanyl-β-alanine, among dipeptide compoundsrepresented by formula (1), or a salt thereof.

Various other objects, features and many of the attendant advantages ofthe present invention will be readily appreciated as the same becomesbetter understood by reference to the following detailed description ofthe preferred embodiments.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In formula (1), an alkyl group represented by R¹ is preferably a C1-C6alkyl group, may be a linear or branched alkyl group, and is morepreferably a methyl group, an ethyl group, an n-propyl group, anisopropyl group, an n-butyl group, an isobutyl group, or a t-butylgroup.

An alkanoyl group represented by R¹ is preferably a C1-C6 alkanoylgroup, may,be a linear or branched alkanoyl group, and is morepreferably an acetyl group, a propionyl group, or a butyryl group.

A lower alkyl group represented by R³, R⁴, R⁶, R⁷, or R⁸ is preferably aC1-C6 alkyl group, may be a linear or branched alkyl group, and is morepreferably a methyl group, an ethyl group, an n-propyl group, anisopropyl group, an n-butyl group, an isobutyl group, or a t-butylgroup.

A lower alkenyl group represented by R⁷ or R⁸ is preferably a C2-C6alkenyl group, more preferably a vinyl group or a propenyl group.

An aralkyl group represented by R⁷ or R⁸ is a C7-C18 aralkyl group.Specific examples include a phenyl C1-C6 alkyl group, a biphenyl C1-C6alkyl group, and a naphthyl C1-C6 alkyl group. Of these, a phenyl C1-C6alkyl group is preferable, and a benzyl group is more preferable.

An alkyl group which may have a substituent, represented by R², may be aC1-C6 alkyl group, a carboxy C1-C6 alkyl group, or a C1-C6alkoxycarbonyl C1-C6 alkyl group. Of these, a C1-C6 alkyl group ispreferable. R² is more preferably a methyl group, an ethyl group, ann-propyl group, an isopropyl group, an isobutyl group, or a t-butylgroup.

An alkyl group which may have a substituent, represented by R⁵, may be aC1-C6 alkyl group, a carboxy C1-C6 alkyl group, or a C1-C6alkoxycarbonyl C1-C6 alkyl group. Of these, a C1-C6 alkyl group and acarboxy C1-C6 alkyl group are preferable. R⁵ is more preferably a methylgroup, an isopropyl group, an isobutyl group, a t-butyl group, acarboxymethyl group, or a carboxyethyl group.

A heterocyclic ring which is formed by R⁴ and R⁵ may be pyrrolidine orpiperidine.

An integer n is preferably 0, 1, or 2.

In formula (1), R¹ is more preferably a hydrogen atom, a C1-C6 alkylgroup, a C1-C6 alkanoyl group, or —CH(R⁶)COOR⁷ (wherein R⁶ represents ahydrogen atom or a C1-C6 alkyl group, and R⁷ represents a hydrogen atom,a C1-C6 alkyl group, a C2-C6 alkenyl group, or a phenyl C1-C6 alkylgroup). R² is more preferably a hydrogen atom or a C1-C6 alkyl group. R³is more preferably a C1-C6 alkyl group or a phenyl group. R⁴ is morepreferably a hydrogen atom or a C1-C6 alkyl group. R⁵ is more preferablya hydrogen atom, a C1-C6 alkyl group, a phenyl C1-C6 alkyl group, or acarboxy C1-C6 alkyl group. X is more preferably —COOR⁸ (wherein R⁸represents a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alkenyl group,or a phenyl C1-C6 alkyl group) or —SO₃H. An integer n is more preferably0, 1, or 2.

Examples of salts of a dipeptide compound (1) include alkali metalsalts, alkaline earth metal salts, amine salts, amino acid salts, andacid addition salts. Of these, alkali metal salts and amino acid saltsare preferable. A dipeptide compound (1) may have optical activity, andits steric configuration may be any of R, S, and a racemic mixture. Thecompound may also be in the form of a hydrate.

Among dipeptide compounds of formula (1), the more preferred compoundsare as follows:

Phe-Gly (compound 11); Phe-β-Ala (compound 12); Phe-Phe (compound 13);Phe-Leu (compound 14); Phe-Ala (compound 15); and Phe-Asp (compound 16):Phe-Tau (compound 17).

Of these, compound 1 and compound 12 are more preferable.

A compound of formula (1) in which R¹ is a hydrogen atom may besynthesized through a process described in “K. Ienalga, K. Higashihara,and H. Kimura, Chem. Pharm. Bull., 35, 1249-1254 (1987).” A compound offormula (1) in which R¹ is not a hydrogen atom but is any of theaforementioned groups may be synthesized through, for example, thefollowing process:

(wherein Y represents a halogen atom, a p-toluenesulfonyl group, or amethanesulfonyloxy group; R¹ represents any of the aforementioned groupsother than a hydrogen atom; and R² through R⁵, n, and X are the same asdescribed above).

Namely, a compound (2) is reacted with a compound (3) in the presence orabsence of a base, and if necessary, hydrolysis by use of a base such assodium hydroxide, or catalytic hydrogenation by use of a catalyst suchas palladium carbon may be: carried out, to thereby yield a compound (1)of the present invention.

Examples of bases which may be employed in the reaction include sodiumhydroxide, potassium hydroxide, potassium carbonate, sodium carbonate,sodium hydrogencarbonate, and triethylamine. Of these, potassiumcarbonate and triethylamine are preferable, and triethylamine is morepreferable.

Examples of solvents which may be employed in the reaction includewater, dimethylformamide, tetrahydrofuran, benzene, toluene, xylene, andhexane. Of these, toluene and benzene are preferable, and toluene ismore preferable. The reaction temperature is −30 to 200° C., preferably10 to 90° C., more preferably 40 to 70° C.

A dipeptide compound (1) or a salt thereof is incorporated into thetopical skin composition of the present invention in an amount of0.0001-40 wt. % (hereinafter “wt. %” will be simply referred to as “%”),preferably 0.01-20%.

The topical skin composition of the present invention is more preferablyused for preventing and ameliorating unfavorable skin conditions causedby aging of the skin, such as wrinkles, flabbiness, and reduction ofelasticity; or for suppressing hair growth.

The topical skin composition of the present invention may furthercontain a keratinization-ameliorating agent, to thereby enhance theeffect of retarding aging of the skin, such as wrinkle formation, or theeffect of suppressing hair growth. Examples of suchkeratinization-ameliorating agents include sphingosine derivatives.

The topical skin composition of the present invention may appropriatelycontain, in addition to the aforementioned ingredients, a variety,ofingredients or carriers which are usually employed in cosmetics,quasidrugs, and drugs. Examples of such ingredients or carriers includehumectants, powders, gelation agents, thickeners, surfactants,emulsifiers, anti-inflammatory agents, antioxidants, pH regulatingagents, chelating agents, preservatives, dyes, perfumes, UVabsorbing-agents, UV protecting agents, existing skin-aging preventiveor retarding agents such as collagen, and existing hair-growthsuppressing agents. The composition may be produced through aconventional method in accordance with the application form.

The topical skin composition of the present invention may be used as askin composition for pharmaceutical use or a cosmetic composition.Examples of pharmaceutical compositions include a-variety of ointmentscontaining a pharmaceutically active ingredient. Such an ointment maycomprise an oily base, or an oil-in-water or water-in-oil emulsion base.No particular limitation is imposed on the species of oily base, and,for example, vegetable oil, animal oil, synthetic oil, fatty acid, ornatural or synthetic glyceride may be employed. No particular limitationis imposed on the species of pharmaceutically active ingredient, and,for example, analgesic and anti-inflammatory agents, antipruritic drugs,astringent agents, or hormones may be appropriately employed inaccordance with needs.

Examples of cosmetic compositions include lotions, emulsions, creams,ointments, sticks, solutions in organic solvents or purified water,packs, gels-and aerosols. Namely, the cosmetic composition may beemployed as a lotion, an oil essence, an O/W-type or, W/O-type cream, apack, a foundation, a skin-cleansing agent, a tonic, a bathing agent, oran aerosol.

EXAMPLES Referential Example 1

Phenylalanyl-β-alanine benzyl ester hydrochloride (10.0 g, 0.028 mol)and benzyl bromoacetate (14.6 g, 0.062 mol) were dissolved intetrahydrofuran (200 mL). To the resultant solution, triethylaminesuspended in tetrahydrofuran was added, and the resultant mixture wasstirred for 42 hours. After completion of the reaction, the resultantmixture was subjected to extraction with ethyl acetate (100 mL), and theorganic layer was washed with a saturated aqueous solution of sodiumhydrogencarbonate and saturated brine and then dried over anhydroussodium sulfate. Subsequently, the solvent was removed, and the resultantresidue was subjected to column chromatography with a solvent mixture ofethyl acetate and n-hexane serving as an eluent. Thereafter, the solventwas removed, to thereby obtain a dibenzyl ester (8.8 g, yield: 66%). Theester was suspended in methanol, and then subjected to catalytichydrogenation by use of palladium carbon (10%). The resultant crystalswere subjected to filtration, and then recrystallized from water, tothereby obtain compound 1 (1.34 g, yield: 35%).

NMR(DMSO-d₆) δ: 2.30(t, 2H, J=7 Hz), 2. 75(dd, 1H, J=8, 14 Hz), 2.87(dd,1H, J=6, 14 Hz), 3.10(dd, 2H, J=17, 25 Hz), 3.22(dt, 2H, J=7,7 Hz),3.33(t, 1H, J=7 Hz), 7.10˜7.36(m, 5H), 7.99(t, 1H, J=6 Hz).

Referential Example 2

Phenylalanyl-γ-aminobutyric acid methyl ester hydrochloride (1.4 g,0.0047 mol) and ethyl bromoacetate (0.8 g, 0.0056 mol) were dissolved intetrahydrofuran (80 mL). To the resultant solution, triethylaminesuspended in tetrahydrofuran was added, and the resultant mixture wasstirred for 24 hours. After completion of the reaction, the resultantmixture was subjected to extraction with ethyl acetate (20 mL), and theorganic layer was washed with a saturated aqueous solution of sodiumhydrogencarbonate and saturated brine, and then dried over anhydroussodium sulfate. Subsequently, the solvent was removed, and the resultantresidue was subjected to column chromatography with a solvent mixture ofethyl acetate and n-hexane serving as an eluent. Thereafter, the solventwas removed, to thereby obtain an ethyl ester (0.5 g, yield: 31%). Theester was dissolved in methanol, and an aqueous solution of sodiumhydroxide (2.2 equivalent) was added to the methanol solution, and theresultant mixture was stirred for 15 hours. Thereafter, the solvent wasremoved, and the resultant residue was neutralized with hydrochloricacid. Then the solvent was removed, to thereby obtain compound 2 (0.18g, yield: 41%).

NMR(DMSO-d₆) δ: 1.52(tt, 2H, J=7, 7 Hz), 2.09(t, 2H, J=8 Hz), 2.86(dd,2H, J=7, 13 Hz), 3.01(dd, 2H, J=8, 14 Hz), 3.10(dt, 2H, J=3, 16 Hz),3.44(t, 1H, J=7 Hz), 7.04˜7.49(m, 5H), 8. 15(t, 1H, J=16 Hz).

Referential Example 3

The procedure of Referential Example 2 was repeated, except thatphenylalanyl-γ-aminobutyric acid methyl ester hydrochloride was replacedby phenylalanyl-leucine methyl ester hydrochloride (1.0 g, 0.00304 mol)and that the amount of ethyl bromoacetate was changed to 0.6 g (0.00365mol), to thereby obtain compound 3 (0.34 g, yield: 33%).

NMR(DMSO-d₆) δ: 0.81(d, 3H, J=6 Hz), 0.86(d, 3H, J=6 Hz), 1.40˜1.68(m,3H), 2.76(dd, 1H, J=8, 14 Hz), 2.93(dd, 1H, J=6, 14 Hz), 3.08(d, 1H,J=17 Hz), 3.23(d, 1H, J=17 Hz), 3.42(dd, 1H, J=6, 8 Hz), 4.16˜4.28(m,1H), 7.17˜7.29(m, 5H), 8.12(d, 1H, J=8 Hz).

Referential Example 4

Phenylalanyl-β-alanine ethyl ester (10 g, 0.038 mol) and triethylamine(10.3 g, 0.1 mol) were suspended in toluene (45 g), and the suspensionwas heated to 60° C. To the resultant suspension, ethyl bromoacetate(9.3 g, 0.057 mol) suspended in toluene (5 g) was added dropwise over0.5 hours, and then the resultant mixture was stirred for three hours.After completion of reaction, the resultant reaction mixture wassubjected to extraction with ethyl acetate (20 g). The organic layer waswashed with a 2.5% aqueous solution of citric acid. Subsequently, thesolvent was removed, to thereby obtainN-(carboethoxymethyl)phenylalanyl-β-alanine ethyl ester (10.7 g, yield:80.3%). The compound was dissolved in methanol, and a 48% aqueoussolution of sodium hydroxide (6.6 g, 0.08 mol) was added to the methanolsolution, and the resultant mixture was stirred for three hours. Aftercompletion of reaction, the solvent was removed, and the resultantresidue was dissolved in water (20 mL), and then washed with ethylacetate (10 g). Subsequently, hydrochloric acid was added to the aqueouslayer, so as to attain a pH of 3.5. After being cooled, the resultantsolution was subjected to filtration, to thereby obtain crude crystals.The crystals were recrystallized from a 20% aqueous solution ofisopropyl alcohol, to thereby obtain compound 1 (6.0 g, yield: 67%).

Referential Example 5

The procedure of Referential Example 4 was repeated, except thatphenylalanyl-μ-alanine ethyl ester was replaced by phenylalanyl-alanineethyl ester (3.7 g, 0.014 mol) and that the amount of ethyl bromoacetatewas changed into 3.5 g (0.021 mol), to thereby obtain compound 4 (1.4 g,yield: 33%).

NMR(DMSO-d₆) δ: 1.24(d, 3H, J=7 Hz), 2.78(dd, 1H, J=8, 14 Hz), 2.96(dd,1H, J=5, 14 Hz), 3.08(d, 1H, J=17 Hz), 3.24(d, 1H, J=17 Hz), 2.46(dd,1H, J=7, 7 Hz), 4.29(dq, 1H, J=7, 7 Hz), 7.13-7.35(m, 5H), 8.22(d, 1H,J=7 Hz), 5.5-8.5(br. s, 2H).

Referential Example 6

The procedure of Referential Example 4 was repeated, except thatphenylalanyl-β-alanine ethyl ester was replaced by phenylalanyl-asparticacid dimethyl ester (7.4 g, 0.024 mol) and that the amount of ethylbromoacetate was changed into 6.0 g (0.036 mol), to thereby obtaincompound 5 (1.0 g, yield: 12%).

NMR(DMSO-d₆) δ: 2.54-2.70(m, 2H), 2.77(dd, 1H, J=7, 14 Hz), 2.93(dd, 1H,J=6, 14 Hz), 3.10(d, 1H, J=17 Hz), 3.28(d, 1H, J=17 Hz), 3.44(dd, 1H,J=6, 7 Hz), 4.45-4.55(m, 1H), 7.19-7.29(m, 5H), 8.28(d, 1H, J=8 Hz).

Referential Example 7

Phenylalanyl-β-alanine (17.4 g, 0.074 mol) was suspended in pyridine(120 g), and the suspension was cooled to 15° C. Acetic anhydride (30 g)was added dropwise to the resultant suspension over one hour. Aftercompletion of addition, the temperature of the resultant mixture wasreturned to room temperature, and the mixture was stirred for 13 hours,and then the reaction was terminated. The resultant reaction mixture wascooled to 5° C., and ethanol (50 mL) was added to the reaction mixture,and the mixture was stirred for 30 minutes, and then the solvent wasremoved. The resultant residue was subjected to extraction with ethylacetate (500 mL), and washed with 5% hydrochloric acid, distilled water,and saturated brine, successively, and then dried over anhydrous sodiumsulfate. Subsequently, the solvent was removed, and the resultantresidue was subjected to column chromatography with a solvent mixture ofchloroform and methanol serving as an eluent. Thereafter, the solventwas removed, to thereby obtain compound 6 (11.5 g, yield: 56%).

NMR(DMSO-d₆) δ: 1.74(s, 3H), 2.30(dd, 2H, J=7, 7 Hz), 2.70(dd, 1H, J=10,14 Hz), 2.91(dd, 1H, J=5, 14 Hz), 3.12-3.36(m, 2H), 4.35-4.46((m, 1H),7.13-7.28(m, 5H), 8.02(t, 1H, J=6 Hz), 8.67(d, 1H, J=8 Hz), 12.22(br. s,1H).

Referential Example 8

Phenylalanyl-β-alanine (1 g, 0.0042 mol) was suspended in water (2 g).To the resultant suspension, palladium carbon (10%) (1 g) and a 36%aqueous solution of formaldehyde (7.1 g, 0.042 mol) were added, and theresultant mixture was stirred in an H₂ atmosphere at 10 atm. for 24hours. After completion of reaction, the solvent was removed. Theresultant residue was purified through column chromatography, to therebyobtain compound 7 (12.8 mg, yield: 8.1%).

NMR(DMSO-d₆) δ: 1.92-2.32(m, 2H), 2.81(s, 6H), 2.89-3.32(m, 2H),3.11(dd, 2H, J=6, 13 Hz), 3.94(dd, 1H, J=4, 10 Hz), 7.00-7.41(m, 5H),8.44(t, 1H, J=6 Hz), 11.82-12.52(br. s, 1H).

Referential Example 9

Phenylalanyl-β-alanine (0.5 g, 0.0021 mol) was suspended in water (5 g).To the resultant suspension, palladium carbon (10%) (0.5 g) andpropionaldehyde (0.15 g, 0.027 mol) in methanol (5 g) were added, andthe resultant mixture was stirred in a H₂ atmosphere for 24 hours. Aftercompletion of reaction, the solvent was removed. The resultant residuewas recrystallized from methanol, to thereby obtain compound 8 (0.13 g,yield: 22.2%).

NMR(DMSO-d₆) δ: 0.75(t, 3H, J=7 Hz), 1.30(ddt, 2H, J=7, 7, 14 Hz),2.12-2.44(m, 2H), 2.28(dd, 2H, J=7, 7 Hz), 2.65(dd, 1H, J=8, 14 Hz),2.81(dd, 1H, J=6, 13 Hz), 3.16(dd, 1H, J=6, 8 Hz), 3.13-3.33(m, 2H),7.05-7.35(m, 5H), 7.89(t, 1H, J=6 Hz).

Referential Example 10

The procedure of Referential Example 4 was repeated, except thatphenylalanyl-β-alanine ethyl ester was replaced by leucyl-β-alanineethyl ester (2.0 g, 0.009 mol) and that the amount of ethyl bromoacetatewas changed into 2.2 g (0.013 mol), to thereby obtain compound 9 (1.6 g,yield: 45%).

NMR(DMSO-d₆) δ: 0.85(d, 6H, J=7 Hz), 1.37(dd, 2H, J=7, 6 Hz),1.48-1.68(m, 1H), 2.37(t, 2H, J=7 Hz), 3.02(d, 1H, J=17 Hz), 3.15(d, 1H,J=17 Hz), 3.27-3.21(m, 3H), 8.26(t, 1H, J=6 Hz).

Referential Example 11

Phenylalanyl taurine (2.5 g, 9.18 mmol) and sodium hydroxide (0.58 g,0.0144 mol) were dissolved in distilled water (25 g), and the solutionwas heated to 50° C. To the resultant solution, bromoacetic acid (1.5 g,0.011 mol) in distilled water (3 g) and an aqueous solution of sodiumhydroxide were added dropwise simultaneously over 0.5 hours, so as toattain a pH of 10.5-11.5, and the resultant mixture was stirred forthree hours. After completion of reaction, concentrated hydrochloricacid was added to the resultant reaction mixture, so as to attain a pHof 3. Thereafter, the solvent was removed, to thereby obtain anN-(carboethoxymethyl)phenylalanyl taurine-inorganic salt mixture (7.15g). The resultant mixture (1 g) was subjected to ODS-gel columnchromatography, to thereby obtain N-(carboethoxymethyl)phenylalanyltaurine (0.15 g). The compound was further treated through fractionationHPLC, to thereby yield compound 10 (48 mg).

NMR (DMSO-d₆) δ: 2.35(dd, 2H, J=7.0, 6.5 Hz), 2.97-3.26(m, 4H), 3.74(d,2H, J=3.3 Hz), 4.01(t, 1H, J=6.7 Hz), 7.19-7.32(m, 5H), 8.29(t, 1H,J=5.5 Hz), 9.41(bs, 1H).

Test Example 1 Suppression of Wrinkle Formation in Hairless Mouse

The back of each hairless mouse (HR/ICR, 6 weeks old at the start of thetest, 5 mice per group) was subjected to a single exposure of UV-B raysat a dose of less than 1 MED using a health light lamp (model: SE20,product of Toshiba Corporation). Immediately after the exposure, an 80%ethanol solution (100 μL) containing a 0.1% test compound was applied tothe back. The above. procedure was carried out daily for 20 weeks. Theradiation energy was measured using a UV-radiometer (model:UVR-305/365D, product of Tokyo Optical). In the control group, only 80%ethanol was applied to the back of each mouse during the test. After thetest, wrinkle formation was visually observed, and the degree of wrinkleformation was evaluated against the following ratings (wrinkle index).The results are shown in Table 1.

(Wrinkle index)

0: No wrinkle formation.

1: A few shallow wrinkles were formed.

2: A slight amount of wrinkles was formed.

3: Some wrinkles were formed.

4: Deep wrinkles were formed.

TABLE 1 Test Compound Concentration Wrinkle Index Control — 3.06 ± 0.13Compound 1 0.1% 1.25 ± 0.11 Compound 2 0.1% 2.01 ± 0.14 Compound 3 0.1%1.82 ± 0.09 Compound 4 0.1% 1.75 ± 0.12 Compound 5 0.1% 2.21 ± 0.14Compound 6 0.1% 2.57 ± 0.20 Compound 7 0.1% 2.34 ± 0.10 Compound 8 0.1%2.03 ± 0.09 Compound 9 0.1% 2.11 ± 0.11 Compound 10 0.1% 1.95 ± 0.12Compound 11 1% 2.82 ± 0.18 Compound 12 1% 1.98 ± 0.20 Compound 13 1%2.64 ± 0.16 Compound 14 1% 2.82 ± 0.22 Compound 15 1% 2.72 ± 0.16Compound 16 1% 2.50 ± 0.15 Compound 17 1% 1.99 ± 0.12

The results shown in Table 1 reveal that dipeptide compounds of formula(1) exhibit excellent effects with respect to suppressing wrinkleformation and retarding aging of the skin or rejuvenating the skin.

Test Example 2 Maintenance of Skin Elasticity in Rat

Three-week-old SD male rats were classified into three groups (five ratsper group): a group of rats to which an 80% ethanol solution containinga test compound was applied to pads of hind limbs; a group of rats towhich a solvent (80% ethanol) was applied to the pads; and a group ofnon-treated rats. Each pad was exposed to UV-B rays (less than 1 MED),and then the test compound-containing solution or the solvent wasapplied to the pad in an amount of 10 μL. The above procedure wascarried out three times a week (every other day of the week days) forsix weeks.

Skin elasticity was determined using a cutometer (model: SES575, productof Courage Kazaka) as follows. The skin of the pad was subjected tosuction at 500 hPa for three seconds, and then released for threeseconds. The displacement over six seconds was measured, to therebyobtain Ue and Uf values. The measurement was repeated five times foreach pad.

The linearity of elastic fibers was analyzed according to the method ofImokawa et al. (J. Invest. Dermatol., 105, 254-258(1995)) through imageanalysis of an SEM micrograph. Specifically, samples for scanningelectron microscopy (.SEM) were prepared by fixing the pad of a rat withMERCOX (product of Dainippon Ink and Chemicals, Inc.) under reflux,followed by digestion with formic acid. From SEM micrographs (×1000) ofeach of the samples, ten typical micrographs were selected and enlargedcopies were made. Each of the enlarged micrographs was divided into 16uniform regions. From each of the regions, any one of the elastic fiberswas selected and traced on a transparent film with a line having a fixedwidth (width: 8 pixels). When the area surrounded by the line tracingthe elastic fiber is called A, and the longitudinal length and laterallength of the minimum rectangle surrounding the trace are called B andC, respectively, the linearity of the elastic fiber is represented byA/(B×C). For example, when the trace of the elastic fiber is linear, thelinearity becomes 1. The results are shown in Table 2.

TABLE 2 Test Compound Concentration Ue value Uf value Linearity Notreatment — 0.03294 ± 0.00251* 0.04833 ± 0.01700* 0.7345 ± 0.0333*Solvent only — 0.02125 ± 0.00875^(#) 0.03333 ± 0.01111^(#) 0.5133 ±0.0600^(#) Compound 1 0.1% 0.03153 ± 0.00523* 0.04711 ± 0.00213* 0.7221± 0.0299* Compound 3 0.1% 0.02985 ± 0.01021* 0.04323 ± 0.01722* 0.7013 ±0.06551* Compound 4 0.1% 0.03009 ± 0.00274* 0.04533 ± 0.00971* 0.7153 ±0.0329* Compound 5 0.1% 0.02694 ± 0.00157* 0.04285 ± 0.00271* 0.6652 ±0.0239* Compound 6 0.1% 0.02433 ± 0.00921* 0.03913 ± 0.00924* 0.6078 ±0.0195* Compound 7 0.1% 0.02623 ± 0.00874* 0.00412 ± 0.00258* 0.6423 ±0.0433* Compound 8 0.1% 0.02708 ± 0.00119* 0.00427 ± 0.00657* 0.6976 ±0.0422* Compound 9 0.1% 0.02793 ± 0.00697* 0.04295 ± 0.00836* 0.6892 ±0.0157* Compound 10 0.1% 0.02972 ± 0.01034* 0.04498 ± 0.00637* 0.7021 ±0.0454* Compound 12   1% 0.03326 ± 0.01267* 0.04572 ± 0.01862* 0.7197 ±0.0321* Compound 17   1% 0.02894 ± 0.00114* 0.04369 ± 0.00872* 0.6913 ±0.0341* *p < 0.05 (vs. solvent only) ^(#)p < 0.05 (vs. no treatment)

The results shown in Table 2 reveal that dipeptide compounds of formula(1) exhibit excellent effects with respect to preventing the lowering ofskin elasticity due to UV-B rays and also preventing degradation of thethree-dimensional structure of the elastic fibers which cause thelowering, and thus the compounds can maintain skin elasticity.

Test Example 3 Hair Growth Suppression in Mouse

The hair on a portion of the backs (size: 2 cm×4 cm) of groups ofsix-week-did C3H mice, each group consisting of five mice, was carefullyshaved so as not to injure the skin using an electric clipper andelectric shaver. To the thus-shaved portion, a sample was applied in anamount of 100 μL twice a day for four weeks. Test compounds weredissolved in a solvent (80% ethanol) to attain the concentrations shownin Table 3, to thereby prepare samples. In the control group, only thesolvent was applied to the shaved portion. After three weeks, in orderto observe hair regrowth, the shaved portion was photographed at acertain magnification, and the ratio of the area of hair regrowth (i.e.,the ratio of the area of hair regrowth to that of the shaved area) wascompared with that of the control group using an image analyzer. Thehair growth suppression ratio (%) was represented by a relative valuewhen the ratio of the area of the hair regrowth in the control group wastaken as 100. The results are shown in Table 3.

TABLE 3 Hair-growth Test compound Concentration suppression ratioCompound 1 0.1% 82.4% Compound 2 0.1% 73.3% Compound 3 0.1% 72.3%Compound 4 0.1% 80.5% Compound 5 0.1% 71.0% Compound 6 0.1% 58.3%Compound 7 0.1% 69.6% Compound 8 0.1% 77.8% Compound 9 0.1% 77.1%Compound 10 0.1% 79.2% Compound 11 1% 32.9% Compound 12 1% 90.4%Compound 13 1% 78.5% Compound 14 1% 70.9% Compound 15 1% 68.4% Compound16 1% 75.2% Compound 17 1% 79.5%

The results shown in Table 3 reveal that dipeptide compounds of formula(1) exhibit excellent effects with respect to suppressing hair growth.

Formulation Example 1

In accordance with the formulation described below, a cream forretarding aging of the skin was produced through a conventional method.The cream exhibited an excellent effect with respect to retarding agingof the skin.

(%) Compound 1 or 12 0.2 Stearic acid 2.0 Cetanol 4.0 Squalene 8.0Vaseline 5.0 Hydrogenated palm oil 4.0 Polyoxyethylene sorbitanmonostearate (20 E.O.) 1.4 Oleophilic glycerin monostearate 2.4 Butylparaben 0.1 Glycerin 3.0 10.0% Potassium hydroxide 0.2 Perfume 0.1Purified water Balance Total 100.0

Formulation Example 2

In accordance with the formulation described below, a pack forpreventing and retarding aging of the skin was produced through aconventional method. The pack exhibited excellent effects with respectto preventing and retarding aging of the skin.

(%) Compound 3 3.0 Polyvinyl alcohol 20.0 Glycerin 5.0 Ethanol 16.0Perfume 0.1 Dye 0.1 Purified water Balance Total 100.0

Formulation Example 3

In accordance with the formulation described below, a hair growthsuppression lotion was produced. Specifically, a solution containingingredients of A and a solution containing ingredients of B wereprepared separately. The solution B was added to the solution A, and theresultant mixture was stirred for homogeneity, to thereby produce a hairgrowth suppression lotion. The lotion exhibited an excellent effect withrespect to suppressing hair growth.

(%) A. Polyoxyethylene hydrogenated castor oil 0.8 Ethanol 30.0 B.Compound 1 or 12 1.0 Sodium dodecyl sulfate 0.12 Dodecylmethylamineoxide 0.18 Isopropyl alcohol 15.0 Benzyl alcohol 12.0 Glycerin 2.0Purified water Balance Total 100.0

Formulation Example 4

In accordance with the formulation described below, a hair growthsuppression aerosol was produced. Specifically, ingredients of A wereuniformly mixed and charged in a container. The container was thenfilled with solution B through a conventional method, to thereby producea hair growth suppression aerosol. The aerosol exhibited an excellenteffect with respect to suppressing hair growth.

(%) A. Compound 1 1.0 Cetanol 1.2 Propylene glycol 4.0 Ethanol 8.0Purified water Balance B. Liquefied petroleum gas (propellant) 4.0 Total100.0

The topical skin composition of the present invention exhibits excellenteffects with respect to retarding aging of the skin or rejuvenating theskin and suppressing hair growth.

What is claimed is:
 1. A method for retarding aging of the skin orrejuvenating the skin comprising applying to a surface of skin in needthereof, a topical skin composition comprising a dipeptide compoundrepresented by formula (1) or a salt of the dipeptide:

wherein R¹ represents a hydrogen atom, an alkyl group, an alkanoylgroup, or —CH(R⁶)COOR⁷, wherein R⁶ represents a hydrogen atom or a C₁₋₆alkyl group, and R⁷ represents a hydrogen atom, a C₁₋₆ alkyl group, aC₂₋₆ alkenyl group, or an aralkyl group; R² represents a hydrogen atomor an alkyl group which may have a substituent; R³ represents a C₁₋₆alkyl group or a phenyl group; R⁴ represents a hydrogen atom or a C₁₋₆alkyl group, and may form a heterocyclic ring together with R⁵ and anadjacent nitrogen atom; R⁵ represents a hydrogen atom, an alkyl groupwhich may have a substituent, or an aralkyl group which may have asubstituent, and may form the heterocyclic ring together with R⁴; Xrepresents —SO₃H or —COOR⁸, wherein R⁸ represents a hydrogen atom, aC₁₋₆ alkyl group, a C₁₋₆ alkenyl group, or an aralkyl group; and n is aninteger of 0-4; and a pharmaceutically acceptable carrier therefor. 2.The method according to claim 1, wherein, in formula (1), R¹ is ahydrogen atom, a C1-C6 alkyl group, a C1-C6 alkanoyl group, or—CH(R⁶)COOR⁷, wherein R⁶ represents a hydrogen atom or a C1-C6 alkylgroup, and R⁷ represents a hydrogen atom, a C1-C6 alkyl group, a C2-C6alkenyl group, or a phenyl C1-C6 alkyl group; R² is a hydrogen atom or aC1-C6 alkyl group; R³ is a C1-C6 alkyl group or a phenyl group; R⁴ is ahydrogen atom or a C1-C6 alkyl group; R⁵ is a hydrogen atom, a C1-C6alkyl group, a phenyl C1-C6 alkyl group, or a carboxy C1-C6 alkyl group;X is —SO₃H or —COOR⁸, wherein R¹ represents a hydrogen atom, a C1-C6alkyl group, a C2-C6 alkenyl group, or a phenyl C1-C6 alkyl group; and nis 0, 1, or2.
 3. The method of claim 1 wherein the dipeptide compoundrepresented by formula (1) is N-(carboxymethyl)phenylalanyl-β-alanine orphenylalanyl-β-alanine.
 4. The method of claim 1, wherein said dipeptidecompound is present in an amount of 0.0001 to 40 wt %.
 5. The method ofclaim 1, wherein said dipeptide compound is present in an amount of 0.01to 20 wt %.
 6. The method of claim 1, wherein said dipeptide is selectedfrom the group consisting of:

Phe-Gly, Phe-β-Ala, Phe-Phe, Phe-Leu, Phe-Ala, Phe-Asp and mixturesthereof.
 7. The method according to claim 1, wherein n=1-4.
 8. Themethod according to claim 1, wherein n=1-2.
 9. The method according toclaim 1, wherein said composition is in the form of an ointment.
 10. Themethod according to claim 1, wherein said composition is selected fromthe group consisting of lotions, emulsions, creams, ointment, sticks,solutions in organic solvents, solutions in purified water, packs, gelsand aerosols.
 11. A method for suppressing hair growth comprisingapplying to a surface of skin in need thereof, a topical skincomposition comprising a dipeptide compound represented by formula (1)or a salt of the dipeptide:

wherein R¹ represents a hydrogen atom, an alkyl group, an alkanoylgroup, or —CH(R⁶)COOR⁷, wherein R⁶ represents a hydrogen atom or a C₁₋₆alkyl group, and R⁷ represents a hydrogen atom, a C₁₋₆ alkyl group, aC₂₋₆ alkenyl group, or an aralkyl group; R² represents a hydrogen atomor an alkyl group which may have a substituent; R³ represents a C₁₋₆alkyl group or a phenyl group; R⁴ represents a hydrogen atom or a C₁₋₆alkyl group, and may form a heterocyclic ring together with R⁵ and anadjacent nitrogen atom; R⁵ represents a hydrogen atom, an alkyl groupwhich may have a substituent, or an aralkyl group which may have asubstituent, and may form the heterocyclic ring together with R⁴; Xrepresents —SO₃H or —COOR⁸, wherein R represents a hydrogen atom, a C₁₋₆alkyl group, a C₁₋₆ alkenyl group, or an aralkyl group; and n is aninteger of 0-4; and a pharmaceutically acceptable carrier therefor. 12.The method according to claim 11, wherein, in formula (1), R¹ is ahydrogen atom, a C1-C6 alkyl group, a C1-C6 alkanoyl group, or—CH(R⁶)COOR⁷, wherein R⁶ represents a hydrogen atom or a C1-C6 alkylgroup, and R⁷ represents a hydrogen atom, a C1-C6 alkyl group, a C2-C6alkenyl group, or a phenyl C1-C6 alkyl group; R² is a hydrogen atom or aC1-C6 alkyl group; R³ is a C1-C6 alkyl group or a phenyl group; R⁴ is ahydrogen atom or a C1-C6 alkyl group; R⁵ is a hydrogen atom, a C1-C6alkyl group, a phenyl C1-C6 alkyl group, or a carboxy C1-C6 alkyl group;X is —SO₃H or —COOR⁸ wherein R⁸ represents a hydrogen atom, a C1-C6alkyl group, a C2-C6 alkenyl group, or a phenyl C1-C6 alkyl group; and nis 0, 1, or
 2. 13. The method of claim 11, wherein the dipeptidecompound represented by formula (1) isN-(carboxymethyl)phenylatanyl-β-alanine or phenylalanyl-β-alanine. 14.The method of claim 11, wherein said dipeptide compound is present in anamount of 0.0001 to 40 wt %.
 15. The method of claim 11, wherein saiddipeptide compound is present in an amount of 0.01 to 20 wt %.
 16. Themethod of claim 11, wherein said dipeptide is selected from the groupconsisting of:

Phe-Gly, Phe-β-Ala, Phe-Phe, Phe-Leu, Phe-Ala, Phe-Asp and mixturesthereof.
 17. The method according to claim 11, wherein n=1-4.
 18. Themethod according to claim 11, wherein n=1-2.
 19. The method according toclaim 11, wherein said composition is in the form of an ointment. 20.The method according to claim 11, wherein said composition is selectedfrom the group consisting of lotions, emulsions, creams, ointment,sticks, solutions in organic solvents, solutions in purified water,packs, gels and aerosols.